The NKG2D receptor expressed by NK cells and activated CDST cells is a potent activator or costimulator of both innate immunity and T cell immunity. The Rae1 and MULT1 ligands for NKG2D are poorly expressed by normal cells but strongly upregulated in virus-infected cells and cancer cells by undefined molecular mechanisms. We have found that expression of Rae1 ligands is induced in normal cells by agents that damage DMA or impart DNA replication stress, but not by other common forms of cell stress. Agents that induce Rae1 activate the DNA damage response, a pathway that plays a central role in maintaining genomic integrity, suppressing tumors and regulating the cell cycle. DNA lesions are recognized by sensor kinases ATM and/or ATR, which activate the checkpoint kinases, Chk2 and Chk1, p53, and ultimately lead to activation of the retinoblastoma protein pRb, cell cycle arrest, and upregulation of DNA repair functions, and, when damage is extreme, apoptosis. We demonstrated that Rae1 upregulation in DNA damaged cells was inhibited by blocking ATM, ATR, or Chk1 using siRNA, conditional gene knockouts or chemical inhibitors. Recently, striking new findings in tumor biology show that human precancerous lesions upregulate the DNA damage pathway and undergo cell cycle arrest, probably triggered by replication stress due to inappropriate proliferation. Advanced tumors, which accumulate many genomic abnormalities, are thought to reactivate the DNA damage pathway. These findings suggested that stable upregulation of Rae1 that occurs in tumor cells may be due to the activity of the DNA damage response. Indeed, we found that inhibiting ATM expression in tumor cell lines with siRNA inhibited Rae1 expression. Other studies show that various viruses activate the DNA damage response when they infect cells. Our hypothesis is that disease-induced genomic insults, via the DNA damage response and Rae1 family molecules, alert the immune system to potential danger arising from the presence of infected, precancerous or cancer cells. The experiments herein are designed to uncover the detailed mechanisms whereby the DNA damage pathway activates expression of NKG2D ligands, determine whether it occurs in developing tumors in vivo, and assess its functional consequences. These data should provide deeper understanding of the role of NK receptors in fighting disease, and may provide the basis for designing potential therapeutic drugs that selectively upregulate Rae1 in diseased cells. Our aims are to: 1) define the proximal regulators of Rae1 genes in the DNA damage pathway; 2) identify elements in the Rae1 gene that regulate gene expression; 3) test the role of the DNA damage response in upregulating Rae1 ligands in developing tumors; 4) determine the functional consequences of the DNA damage pathway-induced Rae1 gene expression; and 5) examine the role of a cellular IRES sequence in regulating MULT1. These studies should advance our understanding of how the immune system surveys cells for loss of genomic integrity and may provide strategies to enhance immunity.